1. Field of the Invention
The present invention relates to the control of the expression of exogenous genes in transgenic animals and in tissue culture cells.
2. Information Disclosure Statement
In agriculture, it is often desirable that an animal have a particular trait. Traditionally, this was accomplished by breeding for the trait. Breeding, unfortunately, has many disadvantages. It may require many generations to fix a desired trait in an animal line. When the bred animals finally acquire that trait, they may also have acquired other, undesired traits. There is no guarantee that a particular individual will acquire the desired trait.
Consequently, a more controlled means of manipulating the phenotype of an animal was sought. The development of recombinant DNA technology offered a possible route to achieving this goal. Theoretically, if a trait was associated with a particular gene that an animal lacked, the gene could be introduced into the animal, thereby modifying its phenotype to include that trait. Of course, there were many hurdles to be surmounted. The genes associated with a particular trait had to be identified and isolated. Suitable regulatory sequences had to be functionally linked to these genes so that they would properly express the trait. The resulting units had to be stably introduced into the cells of the recipient animal. Then, and only then, could the "transgene" function as intended.
The transgene may be expressed in a transgenic animal under the control of a promoter/regulatory domain of choice. The promoter/regulatory domain determines whether the gene is expressed constitutively (at a constant rate and constant level) or whether it is silent or induced depending upon different environmental stimuli. The cytosolic phosphoenolpyruvate carboxykinase (PEPCK) (EC 4.1.1.32) promoter is an example of the latter type of promoter/regulatory domain.
If the exogenous gene is constitutively expressed, the constant expression of the corresponding protein product of this gene may have undesirable effects on the host animal. This could be especially deleterious during embryogenesis when the programmed expression of genes is necessary for orderly development. Consequently, it is desirable to control expression of the gene by means of an inducible or repressible promoter. Additionally, it is desirable to use a promoter which is controllably responsive to changes in diet, since these changes are readily affected.
Many inducible promoters are known. One such promoter regulates the expression of the gene for the cytosolic form of PEPCK, a gluconeogenic enzyme discovered in 1954 by Utter and Kurahashi. This enzyme has a high specific activity in liver, kidney cortex, and white adipose tissue and in lesser levels in lung and jejunum. Hanson and Garber, Am. J. Clin. Nutrition, 25:1010 (1972); Utter and Kurahashi, J. Biol. Chem., 207:287 (1954). There are both cytosolic and mitochondrial forms of PEPCK encoded by different nuclear genes. There are species-specific variations in the expression of both PEPCK forms. The genes for the cytosolic form of this enzyme in the rat and the chicken have been isolated and characterized. Yoo-Warren, et al., PNAS 80:3656-60(1983) (rat) and Hod, Yoo-Warren and Hanson, J. Biol. Chem., 259:15609-15614 (1984) (chicken).
Gluconeogenesis is a process by which non-hexose precursors are converted to glucose to support glucose homeostasis in all vertebrate animals. It occurs only in the liver and kidney cortex. Gluconeogenesis from lactate (Cori cycle) involves 13 enzymes and includes several reactions which also play a role in the citric acid cycle, or in glycolysis, as well as other reactions which are specific for this process. The major precursors for glucose synthesis, in addition to lactic acid, are pyruvic acid, amino acids (such as alanine or glutamine) and glycerol. The pathway is stimulated during periods of starvation or during diabetes, and is depressed by dietary carbohydrate. The major hormonal controls of gluconeogenesis are glucagon (acting via cAMP), which stimulates gluconeogenesis, and insulin, which represses the synthesis of glucose. It is important to distinguish the de novo synthesis of glucose (gluconeogenesis) from glycogenolysis, the breakdown of pre-formed glucose which is stored in the liver and muscle as glycogen. PEPCK is the first committed step in the gluconeogenic pathway and is the pace-setting enzyme in this process. The marked inducibility of the gene for this enzyme reflects the important regulatory position that PEPCK plays in maintaining glucose homeostasis.
In the liver, PEPCK is induced by glucagon, epinephrine, norepinephrine, glucocorticoids, and thyroxine, and deinduced by insulin. In the kidney, acidosis or glucocorticoids elevate PEPCK expression, while alkalosis inhibits PEPCK synthesis. Finally, in adipose tissue, norepinephrine and epinephrine boost PEPCK levels while insulin and glucocorticoids decrease the levels of the enzyme. See Table I, "Factors that alter the levels of PEPCK in rat tissues", in Tilghman, et al., "Hormonal Regulation of Phosphoenolpyruvate Carboxykinase (GTP) in Mammalian Tissues", published as Chapter 2 of Gluconeogenesis: Its Regulation in Mammalian Species, Hanson and Mehlman, eds., (1976).
Hormonal regulation of PEPCK gene expression is tissue-specific. (See Hanson and Mehlman, cited above). However, there is a paucity of information on the sequences responsible for the tissue-specific expression of this gene or for the differences in response to hormones in tissues such as liver and adipose tissue.
Dietary effects on the activity of PEPCK are known. Starvation for 24 hours produces a threefold increase in enzyme activity, which is reversed by a diet high in carbohydrate (e.g., glucose, fructose, and glycerol) or exacerbated by refeeding with a high protein diet. Shrago, et al., J. Biol. Chem., 238:3188 (1963). According to Peret and Chanez, J. Nutrition, 106:103(1976), a high protein diet induced the activity of hepatic PEPCK in mammals (rats), and the activity increased as the protein content of the diet was increased. Pyruvate carboxylase, another gluconeogenic enzyme, was not affected in this manner.
In mammals, the maternal blood supply is cut off at birth, resulting in a transient neonatal hypoglycemia. This results in a fall in the concentration of plasma insulin and a rise in the level of glucagon. This causes an increase in the concentration of hepatic cAMP, which induces the initial expression of PEPCK. The appearance of this enzyme completes the gluconeogenic pathway, and hepatic gluconeogenesis is thereby initiated.
The sequence of the promoter naturally regulating expression of the gene encoding cytosolic PEPCK is given in FIG. 1 of Wynshaw-Boris, et al., J. Biol. Chem., 259:12161 (1984), and is incorporated by reference herein. The rate of transcription of the PEPCK gene in nuclei from the livers of animals induced by hormones is known to be high and is comparable to that reported for the heat-shock gene. See Table 1, in Meissner, et al. (1983), supra. Certain regulatory domains of the "PEPCK promoter" have been identified. Wynshaw-Boris, et al., J. Biol. Chem., 261:9714 (Jul. 25, 1986); Short, et al., J. Biol. Chem., 261:9721 (Jul. 25, 1986). The PEPCK promoter has been used to control expression of both the Herpes virus thymidine kinase (TK) gene and the amino-3'-glycosyl phosphotransferase (AGPT or neo resistance) gene in transfected hepatoma (FTO-2B) cells. Both TK and AGPT synthesis were responsive to cAMP and dexamethasone. Id.; Wynshaw-Boris, et al., BioTechniques, 4(2):104(1986).
Kawasaki, U.S. Pat. No. 4,599,311 advocates the use of yeast promoters which control genes coding for enzymes in the glycolytic pathway (hexokinase 1 and 2, phosphoglucose isomerase, phosphoglycerate kinase, triose phosphate isomerase, phosphoglycerate mutase, pyruvate kinase, phosphofructokinase, enolase, fructose 1, 6-diphosphate aldolase, glyceraldehyde 3-phosphate dehydrogenase, and glycolysis regulation protein). These are coupled to foreign genes and used to control expression of those genes in transformed yeast cells. Kawasaki refers in a general way to regulating the expression by choosing the appropriate nutrient medium. However, he is limited to yeast promoters and yeast cells for the expression of any recombinant gene and production of a given gene product. The gene products may also be inappropriately glycosylated due to the fact that they are secreted by yeast cells. Furthermore, the glycolytic yeast promoters cannot by used in intact animals and will not be expressed in organisms other than yeast.
Kingsman, U.S. Pat. No. 4,615,974 specifically used the yeast phosphoglycerate kinase (PGK) promoter, a glycolytic pathway promoter, to control alpha interferon expression in yeast. Production was induced by introducing glucose into the culture medium.
There is no discussion in either Kawasaki or Kingsman of using diet to control expression of an introduced gene in the cells of a whole animal, or of selecting a gene system which is active essentially only after birth.
Konrad, U.S. Pat. No. 4,499,188 relates to the expression of a heterologous gene in a transformed bacterial cell under TRP promoter control. The medium is initially rich in tryptophan, thereby repressing the gene. Bacterial growth consumes the tryptophan, eventually switching on the gene. The tro promoter is limited to use in prokaryotic cells.
Palmiter, U.S. Pat. No. 4,579,821 describes Herpes virus thymidine kinase (TK) gene expression in adult mice grown from embryos microinjected with a recombinant rDNA vector. This vector contains the TK gene operably linked to the mouse metalothionein-I (MT-I) promoter. This promoter is regulatable by administration of heavy metals such as cadmium or steroid hormones such as dexamethasone. The induction of this promoter or of the mouse MT-II promoter by feeding heavy metals to transgenic animals is inherently limited by considerations of acute and chronic toxicity and teratogenicity. Prolonged feeding of steroid hormones may also have adverse effects. Moreover, since the MT-I promoter, unlike the PEPCK promoter, is active during fetal development, fetal expression of the linked exogenous gene may have deleterious effects upon a transgenic fetus.
While the PEPCK promoter may be induced using dexamethasone, see Wynshaw-Boris, et al. (1986), it is substantially more responsive to hepatic cAMP than to glucocorticoids. Indeed, while liver PEPCK activity may be induced in an adrenalectomized animal by starvation, injection of dexamethasone into a well-fed, adrenalectomized animal does not induce PEPCK activity. Reshef, et al., J. Biol. Chem., 244:5577-81(1969).
The PEPCK promoter is more strongly and rapidly induced by cAMP than is the MT-1 promoter by dexamethasone. In addition, in transgenic animals, the expression of the PEPCK promoter is readily modulated by adjustment of the protein and carbohydrate content of the animal's diet.
There has been considerable interest in using recombinant DNA techniques to express bovine growth hormone or closely-related species, as evidenced by the following references:
Miller, EP Appl 47,600; PA1 Rottman, EP Appl 67,026; PA1 De Boer, EP Appl 75,444; PA1 De Geeter, EP Appl 85,036; PA1 Buell, EP Appl 103,395; PA1 Rottman, EP Appl 112,012; PA1 Aviv, EP Appl 131,843; PA1 Hsiung, EP Appl 159,123; PA1 Kopchick, EP Appl 161,640; PA1 Krivi, EP Appl 193,515; PA1 Aviv, GB 2,073,245; and PA1 Fraser, U.S. Pat. No. 4,443,539.
Particular attention is drawn to Rottman, EP Appl 67,026, which discloses a deposited plasmid (PLG 23) bearing a cDNA copy of the bGH gene, and EP Appl 112,012, setting forth the nucleotide sequence of genomic BGH. None of these references suggest the use of the PEPCK promoter to control bGH expression.